Historically, time-consuming and laborious manual manipulations are part of certain biological assays and lab procedures. Many of these manipulations would be amenable to and benefit from automation. For example, staining of biological material on glass slides requires repeated washing and drying of samples by a scientist or lab technician. Similarly, performing western blot analysis on the bench needs individual attention from a scientist or lab technician, who has to spend hours keeping track of the process and switching solutions manually. In addition, lack of reproducibility and human errors are potential disadvantages of such labor-intensive manual manipulations. It is desirable to replace such manual operations with automation, which would save researchers' valuable time, minimizes operator errors, and increases consistency of experimental results.
Recently automated devices and apparatuses have been developed to automate processing of routine washing and incubation steps, which are required by standard laboratory procedures including western blotting. One example of such automation is the Freedom Rocker™ made by Next Advance. The Freedom Rocker™ is a laboratory platform rocker with a pump and a timer. To operate this apparatus, the user needs to put a membrane, which contains biomolecules to be analyzed, in a tray, place the tray on a shaker platform, store processing reagents in different reservoirs, and automatically dispense reagent into and aspirate reagent from the tray by a pump. The tray is shaken on the shaker platform to ensure sufficient contact between reagents and the membrane in the same tray. One disadvantage of this type of automation is the use of the pump to dispense biological reagents, such as antibody solutions, which could be trapped in the pipelines of the pump and cause contamination or unwanted biological reactions. Another disadvantage is the use of reservoirs to hold biological reagents and the use of tubes to transfer biological reagents from the reservoir to the pump. The dead volume in the reservoirs and tubes would require extra biological reagents to be made available in addition to the amount needed to treat the biological sample in the tray. Certain biological reagents for the bioprocessing, such as antibodies, are either difficult to make or expensive to buy. Therefore, the requirement of extra biological reagents might become a burden for a researcher and a waste of scarce biological reagents.
ProfiBlot™ 48 made by Tecan is another example of western blot processor for automated washing and incubation of strip-based assays. ProfiBlot™ 48 comprises a disposable 48 well tray for sample strips, 3 pumps to dispense and aspirate solutions, 7 reagent bottles and respective delivery tubing, and a control unit. Its operation is similar to that of Freedom Rocker™ made by Next Advance, which involves pumping solutions from reservoirs to each processing container. Therefore, they have similar disadvantages including the use of a pump and the requirement of extra biological reagents.
A third example of automation was disclosed in an international patent application WO2010025302A2 by Life Technology. This patent application WO2010025302A2 described an automated blot processing device. The device stores processing reagents in different reservoirs and allows automatic dispensing and aspiration of bioprocess fluids to a bioprocessing chamber which holds a membrane containing biomolecules to be analyzed. Instead of shaking the sample with fluids, bioprocessing fluids are circulated within the bioprocessing chamber by removing bioprocessing fluids from the chamber via one fluid flow channel and pumping the bioprocessing fluid back to the chamber via a second fluid flow channel. One disadvantage of this device is again the requirement of extra biological reagents due to the employment of reservoirs and tubes to apply biological reagents to the biological sample in the bioprocessing chamber. Another disadvantage is the fixed volume of the bioprocessing chamber for the application of biological reagents and buffers. A smaller volume for the application of biological reagents than that for the application of buffers would decrease the required amount of scarce biological reagents, such as antibodies. The third disadvantage is that the size of the processing chamber limits the size of the membrane to be analyzed.
A fourth example of automated processor for western blot is BlotCycler™ made by Precision Biosystems. The device automatically dispenses and aspirates reagents from multiple trays simultaneously. A tank stores all the washing buffers while smaller chambers contain different antibodies. A fluid distribution technology is used to deliver buffers and antibodies to each processing tray and a shaking mechanism is used during the incubation. Similar to the aforementioned automations, a membrane stays in the same tray during the process.
In all four examples the membrane to be analyzed stays in the same container, be it a tray, a well or a chamber, throughout the whole process. Unwanted mixing of different bioprocessing fluids might occur if part of a previous bioprocessing fluid is trapped in the container when a different bioprocessing fluid is dispersed into the same container. In addition, a large amount of antibodies is needed for the automation since the antibody solutions need to cover the whole surface of the membrane to be processed.
There is still a need in the art for simple, affordable, and easy to use apparatus which allows for automated processing of biological samples. Due to the high cost and limited amount of the biological reagents used as processing fluids in staining and western blot, it would be advantageous to reduce the volume of biological reagents applied without reducing the concentration thereof. One way to accomplish this goal is to add the biological reagents directly to the bioprocessing container without employing extra reservoirs and transferring tubes to hold the biological reagents. Another way is to reduce the volume of the bioprocessing container while maintaining the even distribution of processing fluids over the surface of the membrane. Moreover, conducting different steps of the blotting process in different bioprocessing containers might reduce the chance and the level of contaminations during the process. The invention described here addresses these and related needs.